Tuberculosis (TB) is responsible for causing 5 million deaths annually. The presence of increased number of people having double infections with MTB and human immunodeficiency virus emphasizes the importance of controlling this infection. The major problem in treating the disease lies in the fact that most of the infections are asymptomatic and latent. About 3 billion world population is infected with latent form of TB which if left untreated, kills more than 50% of the infected people. Further, the treatment of TB requires administration of multiple antibiotics over a long period of time. This leads to development of multiple drug-resistant tuberculosis (MDR-TB) infection aggravating the problems of TB treatment.
The World Health Organization has therefore declared as a priority the need to immediately control tuberculosis infection to prevent the spread of drug-resistant strains.
Infection of a mammalian host by M. tuberculosis usually occurs by the aerosol route, and the macrophages in the lung are typically affected. Macrophages are among the most important players in the characteristic immune defenses that control different infectious processes. Non-replicating M. tuberculosis bacilli under in vitro culture conditions are characteristically resistant to most of the anti-tubercular agents and usually known as dormant bacilli. It is documented that non-pulmonary tissue oxygen concentrations within the human body are well below the oxygen concentration in ambient room air. Furthermore, the oxygen concentration in the phagosome of activated macrophages is lower than the extracellular oxygen concentration. The MTB cells within lipid-loaded macrophages lose acid-fast staining, becoming phenotypically resistant to the two frontline anti-mycobacterial drugs rifampicin and isoniazid, and induce gene transcripts involved in dormancy and lipid metabolism within the pathogen. The pathogen thus acquires the phenotypically drug-resistant non-replicating state during latent infection and creates major hindrance to curing the disease. Hence, humans harboring latent tuberculosis infection (LTBI) carry a lifetime risk of reactivation to active disease.
Early detection of TB is therefore of paramount importance in curing this fatal infection. A definitive diagnosis of tuberculosis can only be made by culturing Mycobacterium tuberculosis organisms from a specimen taken from the patient (most often sputum, but may also include pus, CSF, biopsied tissue, etc. (Virtanen S. (1960). Acta Tuberc. Scand. 47: 1-116). A diagnosis made other than by culture may only be classified as “probable” or “presumed”. For a diagnosis negating the possibility of tuberculosis infection, most protocols require that two separate cultures both test negative (Virtanen S. (1960). Acta Tuberc. Scand. 47: 1-116). A complete medical evaluation for TB must include a medical history, a physical examination, a chest X-ray and microbiological examination (of sputum or some other appropriate sample). It may also include a tuberculin skin test, other scans and X-rays, surgical biopsy. A physical examination is done to assess the patient's general health and find other factors which may affect the TB treatment plan. It cannot be used to confirm or rule out TB. Certain cases require a specimen that cannot be supplied by sputum culture or bronchoscopy. In these cases, a biopsy of tissue from the suspected system can be obtained by mediastinoscopy.
Interferon-γ (interferon-gamma) release assays (IGRAs) are based on the ability of the Mycobacterium tuberculosis antigens for early secretary antigen target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) to stimulate host production of interferon-gamma. Because these antigens are not present in non-tuberculous mycobacteria or in BCG vaccine, these tests can distinguish latent tuberculosis infection (LTBI). The blood tests QuantiFERON-TB Gold and T-SPOT.TB use these antigens to detect people with tuberculosis. Lymphocytes from the patient's blood are cultured with the antigens. These tests are called interferon γ tests and are not equivalent. If the patient has been exposed to tuberculosis before, T lymphocytes produce interferon γ in response. Both tests use ELISA to detect the interferon γ with great sensitivity. The distinction between the tests is that QuantiFERON-TB Gold quantifies the total amount of interferon γ when whole blood is exposed to the antigens, whereas T-SPOT.TB, a type of ELISPOT assay, counts the number of activated T lymphocytes that secrete interferon γ. Guidelines for the use of the FDA approved QuantiFERON-TB Gold were released by the CDC in December 2005. The overall purpose of the present invention is to find a diagnostic tool to assess the presence of dormant tubercle bacilli in humans. Our diagnostic procedure mainly relies on the sensitivity towards tubercular metabolite. Major problem of current tuberculosis treatment lies also in its inability to assess the conversion of active to dormant bacilli in humans. Drug induced dormant bacilli is not killed by currently available drugs and is also the major reason for resistance and long treatment period.
There is no diagnostic method available to check the increase or decrease of bacilli after the treatment has started. Available techniques rely on either immunological or staining technique or culturing of actively growing bacilli. These methods work particularly at a very initial stage when the patient is critically ill and has not just started taking medicine. It becomes ineffective within few weeks of starting the treatment. Major problem lies in the failure to detect the bacilli present in dormant stage and not effectively killed by medicine. In most of the hospitals, the methods followed on a regular basis do not detect the bacilli with surety. As a result, patients are compelled to follow wrong diagnosis.
Moreover, identification of the intracellular target of a lead inhibitor is imperative for pursuing the requisite lead program.
Wayne's hypoxia and nutrient starvation-induced dormancy models were developed to explain certain features in persistent tubercular bacilli obtained from hosts. Pioneering work by Wayne showed that nitrate reductase (NarGHJI) played an important role during transition from the aerobic to anaerobic dormant stage and that this transition occurs during initial exposure to the asymptomatic pathogenesis as well as during exposure to anti-tubercular medicines.
Recent reports suggest that nitric oxide (NO) and superoxide (O2−) are generated inside host macrophages and kill the intracellular bacilli after infection by combining to form highly unstable peroxynitrite (ONOO−), which subsequently rearranges to produce NO3− (Nyka, W. studies on the effect of starvation on Mycobacteria. Infect. Immun. 1973, 843-850) that can act as a source of nitrogen or as an alternate electron acceptor during hypoxia-induced dormancy in absence of oxygen. These findings indicate that nitrate metabolic pathway plays a crucial role in Mycobacterium tuberculosis survival under dormancy.
Article titled “Nitrate reduction as a marker for hypoxic shiftdown of Mycobacterium tuberculosis” by L. G. Wayne, L. G. Hayes in Journal: Tubercle and Lung Disease—TUBERCLE LUNG DIS″, Vol. 79, no. 2, pp. 127-132, 1998, DOI: 10.1054/tuld.1998.0015 characterizes nitrate reduction during aerobic growth and hypoxic shift down to non-replicating persistence of Mycobacterium tuberculosis cultures.
Article titled “Bactericidal activity of 2-nitroimidazole against the active replicating stage of Mycobacterium bovis BCG and Mycobacterium tuberculosis with intracellular efficacy in THP-1 macrophages” by Arshad Khan, Sampa Sarkar, Dhiman Sarkar in International Journal of Antimicrobial Agents, Volume 32, Issue 1, July 2008, Pages 40-45 evaluates the anti-tubercularous potential of 2-nitroimidazole, under in vitro conditions, against M. tuberculosis in the intracellular environment of the human monocytic cell line THP-1.
Moreover, Glutamine synthetase (GS), another enzyme in nitrate metabolic pathway is known to convert ammonia and glutamate to glutamine, which is the only known pathway for ammonia utilization in MTB and is observed to be essential for the synthesis of poly L-glutamate/glutamine in cell wall formation.

Nitrate reduction is found to be induced during transition of actively growing cells into hypoxic condition. Nitrate is used as alternate respiratory substrate for accepting electrons in absence of O2 in the medium; however, no physiological investigation has been carried out on nitrite reductase that provides a metabolic link between nitrate reductase (NarGHJI) and glutamine synthetase (GS) which can help in understanding the role of nitrate metabolism during dormancy. So far, GS is reported to play an essential role in growing aerobic bacilli. Its role in dormant bacilli is unknown. Host macrophages are reported to produce NO and superoxide to kill intracellular bacilli, which is supposed to combine and rearrange to produce nitrate inside infected macrophages. Unless hypoxia is achieved, this nitrate is not required either as respiratory substrate or as nitrogen source for survival within host environment. In fact, the functional role of nitrate metabolic pathway of Mycobacterium tuberculosis during survival within host macrophages has remained unexplored so far. The reported estimates of net nitrate synthesis by mammalian tissue vary greatly and range from 0.15 to 1 mM day-1 (Kelm M. (1999) Biochim. Biophys. Acta 1411: 273-289). Within the tissue, nitrate is mainly a product of spontaneous degradation of nitric oxide. Nitric oxide, in contrast, is produced enzymatically by three different nitric oxide synthetases (Stuehr D J. 1999. Biochim. Biophys. Acta 1411: 217-230). An inducible nitric oxide synthetase is expressed in response to inflammatory and proinflammatory mediators (Bogdan C, et. al. (2000) Curr. Opin. Immunol. 12: 64-76). A variety of cells, including hepatocytes, can be induced to synthesize nitric oxide (Brown G C. (1999) Biochim. Biophys. Acta 1411:351-369). Significant amounts of nitrate are detected in the urine of mice infected with bacteria, suggesting that nitrate is available in the kidney, especially in animals undergoing an inflammatory process (Flesch I E, et. Al/ (1991) Infect. Immun. 59: 3213-3218.). Although nitrite in urine samples is being used for the detection of UTI infection, there is no report of using either nitrate or nitrite in the detection of TB.
The latent tubercle bacilli are broadly suggested to be present in granulomas structures within infected humans. Surprisingly, Cornell model of drug induced dormancy suggests exposure of bacilli within animal host induces latency and these cells reactivate to the actively growing stage as soon as the drugs are withdrawn. It is well known that in presence of even trace amount of nitrate/nitrite, survival of bacilli becomes dependent on their use as alternate electron acceptor under hypoxic conditions. But the question still remains in the aspect of Mycobacterium tuberculosis survival during its transition from actively growing to dormant stage with the help of this very weak supply of nitrate at an inaccessible place within our body. At this stage it is unexpected that detection of Mycobacterium tuberculosis cells would be possible from still lower conversion of this low level of nitrate in body fluids. The question also arises as to how these latent bacilli are reactivated so easily but not the latent bacilli residing within our body under normal conditions. Apart from addressing the problem associated with latency of the bacilli as well as the failure of current screening methods in identifying latent stage specific anti-mycobacterial drugs is a pressing need to provide a drug target which could additionally function as biomarker for the identification of active and dormant stage inhibitors of Mycobacterium tuberculosis. 